Application of traditional Chinese medicine composition in preparation of medicine for preventing and / or treating ovarian dysfunction

Abstract

The invention provides application of a traditional Chinese medicine composition in preparation of a medicine for preventing and / or treating ovarian dysfunction, and belongs to the technical field of biological medicine. The traditional Chinese medicine composition comprises the following traditional Chinese medicine raw materials in parts by weight: 8-10 parts of American ginseng, 2-4 parts of pseudo-ginseng, 55-65 parts of kudzuvine root, 42-48 parts of coptis chinensis, 42-48 parts of scutellaria baicalensis, 14-16 parts of stiff silkworm, 14-16 parts of rhizoma zingiberis, 42-48 parts of bitter gourd and 55-65 parts of rhizoma anemarrhenae. The traditional Chinese medicine composition has the effects of increasing the number of follicles and corpus luteum, restoring the duration of an estrus cycle and restoring the levels of serum hormones and sex hormone receptors. The traditional Chinese medicine composition provided by the invention provides a new means for preventing and treating ovarian dysfunction.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a traditional Chinese medicine composition in preparation of a medicine for preventing and / or treating ovarian function disorder. BACKGROUND

[0002] The ovary is the female gonad, and has two functions of reproduction and endocrine. The ovarian function disorder refers to abnormality of the ability of producing ovum and secreting hormone, thereby causing problems such as irregular menstruation and decreased fertility. The most common hormone disorder is the increase of serum testosterone (T) and luteinizing hormone (LH), which is manifested as reduced menstrual flow, early menopause, abnormal hormone level, reduced total number of follicles, and the like. In severe cases, the female can be infertile.

[0003] At present, common drug treatment methods for the ovarian function disorder include administration of estrogen and progesterone, ovulation induction treatment and traditional Chinese medicine treatment. Common traditional Chinese medicines for treating ovarian function decline include Kuntai Capsules, Dingkun Dan, Wujibaifeng Pills, Compound Colla Syrup, Zuogui Pills and the like. At present, the treatment of ovarian disorders by traditional Chinese medicine has the problem of poor treatment effect. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a traditional Chinese medicine composition which can treat the ovarian function disorder by increasing the number of follicles and corpus luteum, restoring the length of the estrus cycle, and restoring the levels of each serum hormone and sex hormone receptor.

[0005] The present application provides a traditional Chinese medicine composition comprising the following traditional Chinese medicine raw materials by weight: American ginseng 8-10 parts, Sanchi 2-4 parts, Pueraria 55-65 parts, Coptis 42-48 parts, Scutellaria 42-48 parts, Scolithus 14-16 parts, Dried ginger 14-16 parts, Bitter gourd 42-48 parts and Anemarrhena 55-65 parts.

[0006] Preferably, the traditional Chinese medicine composition comprises the following traditional Chinese medicine raw materials by weight: American ginseng 9 parts, Sanchi 3 parts, Pueraria 60 parts, Coptis 45 parts, Scutellaria 45 parts, Scolithus 15 parts, Dried ginger 15 parts, Bitter gourd 45 parts and Anemarrhena 60 parts.

[0007] The present application provides a preparation method of the traditional Chinese medicine composition, comprising the following steps:

[0008] Water extraction of American ginseng, Sanchi, Pueraria, Coptis, Scutellaria, Scolithus, Dried ginger, Bitter gourd and Anemarrhena at 70-90℃ to obtain an extract;

[0009] Concentration of the extract and purification with an alcohol solution with a volume percentage of 35%-45% to collect the supernatant and remove the alcohol solvent to obtain the traditional Chinese medicine composition.

[0010] Preferably, the solid-liquid ratio is 1:8-1:12 during the water extraction or alcohol extraction.

[0011] The time of the water extraction or alcohol extraction is 1-2 hours, and the number of times of the water extraction or alcohol extraction is 2-3 times;

[0012] The time of the ultrasonic-assisted extraction is 30-60 minutes, and the frequency of the ultrasonic-assisted extraction is 40-60 kHz;

[0013] The concentration includes concentration under reduced pressure, and the concentration is to obtain a clear extract with a relative density of 1.05-1.15 at 60℃;

[0014] When the alcohol purification is performed, the working volume percentage of the alcohol is 70%-80%.

[0015] The application provides application of the traditional Chinese medicine composition prepared by the preparation method in preparation of a medicine for preventing and / or treating ovarian dysfunction.

[0016] Preferably, the ovarian dysfunction includes at least one of the following: menstrual cycle disorder, ovarian mass reduction, reduction in the number of primary follicles and mature follicles, reduction in the number of corpus luteum, hormone disorder and sex hormone receptor increase.

[0017] Preferably, the hormone disorder includes at least one of the following: reduction in anti-Mullerian hormone (AMH) and follicle-stimulating hormone (FSH), increase in serum testosterone (T) and increase in luteinizing hormone (LH).

[0018] Preferably, in the medicine, the concentration of the traditional Chinese medicine composition is 0.5 g / g or more.

[0019] The application provides a medicine for treating ovarian dysfunction, and the active ingredient includes the traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the preparation method.

[0020] The application provides a traditional Chinese medicine composition, which comprises the following traditional Chinese medicine raw materials in parts by weight: American ginseng 8-10 parts, Panax notoginseng 2-4 parts, Pueraria 55-65 parts, Coptis 42-48 parts, Scutellaria 42-48 parts, Scolithus 14-16 parts, dried ginger 14-16 parts, bitter gourd 42-48 parts and Anemarrhena 55-65 parts. The Pueraria, Coptis and Scutellaria in the traditional Chinese medicine composition play the pharmacological effects of clearing heat and drying dampness, activating the spleen and generating fluid; the Anemarrhena plays the effect of nourishing yin and moistening dryness; the bitter gourd plays the effect of benefiting qi, invigorating the spleen and tonifying the kidney; the American ginseng plays the effect of nourishing yin and generating fluid; the Panax notoginseng plays the effect of removing blood stasis and stopping bleeding; the Scolithus plays the effect of anti-inflammation and immune regulation; and the dried ginger plays the effect of warming the middle and dispelling cold, assisting yang and transforming qi. The application takes a mouse model with T2DM, glucose and lipid metabolism disorder and ovary function disorder as the experimental object, and administers the traditional Chinese medicine composition in the form of oral dosage. The results show that, compared with the model group, the treatment of the traditional Chinese medicine composition with different dosages can restore the estrus cycle of the model mice to different degrees, increase the levels of AMH and FSH of the mice, reduce the increase of the level of serum testosterone (T) and luteinizing hormone (LH), and down-regulate the expression levels of the mRNA of CYP19A1 and LHR; in addition, the traditional Chinese medicine composition can also improve the ovary appearance, ovary index and follicle number of the model mice. Specifically, compared with the decreased ovary quality of the model mice, the administration of the traditional Chinese medicine composition can improve the reduction of the ovary quality; the H&E staining results show that, compared with the reduced number of each ovary of the model mice, the administration of the traditional Chinese medicine composition can increase the number of primary follicles and mature follicles, reduce the number of atretic follicles, and significantly increase the percentage of mature follicles. It can be seen that the traditional Chinese medicine composition provided by the application can prolong the estrus cycle of the model mice, improve the ovary appearance, ovary index and follicle number, restore the serum hormone level and reduce the expression level of sex hormone receptors, so as to achieve the treatment purpose of ovary function disorder. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The verification results of the ovary function impaired mouse model, wherein A is the ovary quality result of the mice; B is the H&E staining result; C is the primary follicle number result; D is the secondary follicle number; E is the mature follicle number result; F is the corpus luteum number result; G is the atretic follicle number result; H is the mature follicle percentage result;

[0022] Figure 2Effects of the Chinese medicine compound on the estrous cycle, serum hormones and sex hormone receptors of model mice, wherein A-D are typical staining results of vaginal smears at different stages, E-K are fold line graphs of the estrous cycle of mice in different groups, L is a stacked column chart of the proportion of each phase of the estrous cycle of mice in different groups, M is the serum AMH concentration result, N is the serum T concentration result, O is the serum FSH concentration result, P is the serum LH concentration result, Q is the serum E concentration result, R is the relative expression amount result of CYP19A1 at the mRNA level, S is the relative expression amount result of FSHR at the mRNA level, and T is the relative expression amount result of LHR at the mRNA level, n = 3-6, * compared with the Con group, p < 0.05, # compared with the Model group, p < 0.05;

[0023] Figure 3 Effects of the Chinese medicine compound on the appearance of ovaries, ovarian index and follicle number of model mice, wherein A is the size result of the ovaries of mice, B is the weight result of the ovaries of mice, C is the ovarian index result of mice, D is the primary follicle number result, E is the secondary follicle number result, F is the natural follicle number result, G is the corpus luteum number result, H is the atretic follicle number result, I is the mature follicle proportion result, and J is the H&E staining result. DETAILED DESCRIPTION

[0024] The present application provides a Chinese medicine composition, which comprises the following Chinese medicine raw materials by weight: American ginseng 8-10 parts, Sanchi 2-4 parts, Gegen 55-65 parts, Huanglian 42-48 parts, Huangqi 42-48 parts, Jieseng 14-16 parts, Ganjiang 14-16 parts, Kugua 42-48 parts and Zhimu 55-65 parts.

[0025] In the present application, the Chinese medicine composition preferably comprises the following Chinese medicine raw materials by weight: American ginseng 9 parts, Sanchi 3 parts, Gegen 60 parts, Huanglian 45 parts, Huangqi 45 parts, Jieseng 15 parts, Ganjiang 15 parts, Kugua 45 parts and Zhimu 60 parts.

[0026] In the present application, Huanglian is used in a large amount in the Chinese medicine composition to clear heat and dry dampness, and it is recorded in Shennong Bencaojing that the medicine has the function of stopping diabetes, Huangqi is used in a large amount to clear heat and purge fire, both of which enter the stomach, large intestine and liver meridian and are used together as the monarch drugs; Kugua clears heat and resolves toxicity, Gegen generates fluid and stops thirst, Zhimu nourishes yin and moistens dryness, American ginseng supplements qi and nourishes yin, and the above four drugs are used together as the minister drugs to enhance the function of clearing heat and purging fire and at the same time take into account the damage of heat to yin; Ganjiang warms the middle and dispels cold, and it is used as an auxiliary drug to neutralize the damage of cold and cool drugs to the spleen and stomach; Sanchi disperses blood stasis and stops bleeding and dredges blood vessels, and Jieseng resolves phlegm and dispels nodules, and modern pharmacology has confirmed that Jieseng has the function of reducing blood sugar, and both of them are used together as the minister drugs. The Chinese medicine composition plays a synergistic effect in preventing and treating ovarian function disorder.

[0027] The present application does not have special restrictions on the source of the traditional Chinese medicine raw materials, and the traditional Chinese medicine raw materials known in the art can be used. In the examples of the present application, each traditional Chinese medicine raw material is purchased from Ningxia Mingde Pharmaceutical Co., Ltd.

[0028] The present application provides a preparation method of the traditional Chinese medicine composition, comprising the following steps:

[0029] The American ginseng, panax notoginseng, radix puerariae, rhizoma coptidis, radix scrophulariae, silkworm chrysalis, dried ginger, bitter gourd and radix anemarrhenae are water-extracted at 70-90℃ to obtain an extract;

[0030] The extract is concentrated and then purified with an alcohol solution with a volume percentage of 35%-45%, the supernatant is collected, the alcohol solvent is removed, and a traditional Chinese medicine composition is obtained.

[0031] The present application water-extracts the American ginseng, panax notoginseng, radix puerariae, rhizoma coptidis, radix scrophulariae, silkworm chrysalis, dried ginger, bitter gourd and radix anemarrhenae at 70-90℃ to obtain a first extract and a residue.

[0032] In the present application, the American ginseng, panax notoginseng, radix puerariae, rhizoma coptidis, radix scrophulariae, silkworm chrysalis, dried ginger, bitter gourd and radix anemarrhenae are preferably pulverized before water extraction or alcohol extraction so as to fully extract active pharmaceutical ingredients. The particle size of the pulverized raw materials is preferably 80-100 mesh, and can be 90 mesh.

[0033] In the present application, the solid-liquid ratio is preferably 1:8-1:12, and can be 1:10, during water extraction. The water extraction time is preferably 1-2h, and can be 1.5h. The water extraction is preferably performed 2-3 times. The two extracts are combined. The water extraction temperature is preferably 75-85℃, and can be 80℃. The extraction temperature is conducive to improving the extraction efficiency of berberine and baicalin in rhizoma coptidis and radix scrophulariae.

[0034] After obtaining the extract, the present application concentrates the extract and then purifies it with an alcohol solution with a volume percentage of 40% to obtain a traditional Chinese medicine composition.

[0035] In the present application, the concentration preferably comprises concentration under reduced pressure. The concentration is preferably performed to obtain a clear extract with a relative density of 1.00-1.05 at 60℃. The method of alcohol purification preferably comprises adding alcohol to the dissolved clear extract, standing, filtering, and collecting the precipitate. The working volume percentage of the alcohol is preferably 70%-80%, and can be 75%. The standing time is preferably 24-48h, and can be 36h. The precipitate preferably comprises drying to obtain a traditional Chinese medicine composition. The drying preferably comprises spray drying or vacuum freeze drying. The inlet air temperature of the spray drying is preferably 180-200℃, and the outlet air temperature is preferably 80-100℃, which is conducive to retaining volatile components.

[0036] In the present application, the preparation method preferably comprises extracting the traditional Chinese medicine raw material with 10 times the volume of water for 100 min, extracting twice, combining the two times of extracted liquid, and concentrating to a relative density of 1. After cooling to room temperature, alcohol (ethanol) is added to the medicinal liquid to a content of 40%, alcohol precipitation is performed for 24 h, concentration is performed until no alcohol taste is present, drying is performed, and powder is prepared. The drying comprises baking or reduced pressure drying. The temperature of the baking is preferably 55-65°C, and can be 60°C. The time of the baking is preferably 2.5-3.5 h, and can be 3 h. The temperature of the reduced pressure drying is preferably 70-75°C. The time of the reduced pressure drying is preferably until constant weight.

[0037] In an embodiment of the present application, the preparation method comprises extracting 297 g of traditional Chinese medicine with 10 times the volume of water for 100 min, extracting twice, concentrating the two times of extracted liquid to 297 mL, cooling to room temperature, adding industrial alcohol to a content of 40% (297 mL of medicinal liquid concentrate + 198 mL of ethanol), alcohol precipitation for 24 h, filtering with gauze folded four times, concentrating, placing in an oven at 60°C for 3 h or so, grinding, and preparing powder.

[0038] The preparation method further comprises weighing 297 g of the raw material of the traditional Chinese medicine composition according to the prescription amount, adding 10 times the amount of water to decoct and extract twice, each time for 100 min, filtering the decocted liquid, combining the filtrate and concentrating to a relative density of 1.00, cooling to room temperature, slowly adding 95% ethanol to a content of 40% while stirring, sealing and placing in a cool place, taking the supernatant after 24 h, concentrating at 40°C under reduced pressure until no alcohol taste is present, drying at 70-75°C under reduced pressure to form a concentrated solution, placing the concentrated solution in an evaporating dish dried to constant weight in a water bath at 80°C, and evaporating to a flow extract, for standby use.

[0039] The present application provides a medicine for treating ovarian dysfunction, and the active ingredient comprises the traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the preparation method.

[0040] In the present application, the medicine preferably further comprises a pharmaceutically acceptable excipient. The excipient comprises at least one of the following: a filler, a binder, a disintegrant, a lubricant, a glidant, a wetting agent, an effervescent agent, a coloring agent, a sweetening agent, a preservative, a dispersing agent, a film-forming agent, a film-forming agent, a plasticizer, a pore-forming agent, an opacifying agent, a pH adjusting agent, an osmotic pressure adjusting agent, an integrating agent, a penetration enhancer, a thickening agent, a humectant, a propellant, and a controlled-release material. The filler, also known as a diluent, is used to fill the volume and weight of the tablet or capsule, thereby diluting the main drug and facilitating tabletting and filling. The binder can increase the mechanical strength of the tablet, enabling it to maintain its overall shape better. The disintegrant can cause the drug to rapidly disintegrate and dissolve in the gastrointestinal tract, allowing for better absorption of the drug. The lubricant can reduce friction between drug particles, making the formulation flow more easily. The glidant can increase the flowability of the formulation, improving the uniformity and compressibility of the formulation. The wetting agent can allow solid drugs to better contact with liquids, promoting dissolution and absorption. The effervescent agent can generate gas bubbles in the body, promoting the dissolution and release of the drug. The coloring agent can impart a specific color to the drug, allowing for identification and differentiation of different formulations. The sweetening agent improves the taste of the drug. The preservative protects the chemical structure of the drug, extending the shelf life of the drug. The dispersing agent improves the dispersibility of the drug. The film-forming agent is used to make film-controlled release formulations. The plasticizer increases the flexibility and plasticity of the material. The pore-forming agent is used to prepare porous matrix tablets to control drug release. The opacifying agent prevents the effects of light on the drug. The pH adjusting agent adjusts the pH of the formulation to adapt to the optimal release conditions of the drug or to improve patient compliance. The osmotic pressure adjusting agent is used to adjust the osmotic pressure of the formulation to prevent hemolysis or affect drug absorption. The integrating agent is used to stabilize the drug solution, preventing drug molecules from aggregating. The penetration enhancer is used to enhance the ability of the drug to penetrate biological membranes, improving the bioavailability of the drug. The thickening agent is used to increase the viscosity of the formulation, controlling the release rate of the drug. The humectant is used to maintain the moisture of the formulation, preventing the drug from failing due to water loss. The propellant is used for aerosol and powder inhalation, providing power to spray the drug out. The controlled-release material is used to prepare sustained-release or controlled-release formulations to control the release rate of the drug.

[0041] In the present application, the dosage form of the medicine preferably includes oral dosage forms, which can specifically include at least one of the following: tablets, powders, granules, capsules, and oral liquids, etc. In the embodiments of the present application, in order to verify the efficacy of the traditional Chinese medicine composition, the traditional Chinese medicine composition is prepared into granules for oral administration, but this cannot be understood as a limitation on the scope of protection of the present application. The present application does not have special limitations on the preparation method of the oral dosage forms, and the preparation method of the oral dosage forms known in the art can be used.

[0042] In the present application, the preparation method of the granules is as follows: the above prepared traditional Chinese medicine composition powder is mixed with excipients, placed in a V-type mixer and dry mixed until uniform, then the mixed powder is directly compressed into a sheet by a roller compactor and broken into granules. The excipients include dextrin and sucrose powder or dextrin and β-cyclodextrin. The dextrin acts as a binder and is used in an amount of 10% to 20% of the mass of the powder, which can be 15%. The sucrose powder is added in an amount of 20% to 30% of the mass of the powder, which can be 25%. The β-cyclodextrin is a bitter taste inclusion excipient and is used as a filler / flavoring agent in an amount of 20% to 30% of the mass of the powder, which can be 25%. The dry granulation is preferably carried out by a microwave-assisted granulation process, specifically, the residual 5% to 8% water in the powder is heated to 60 to 80°C by microwave to promote water vaporization and realize powder bonding, so that granulation can be achieved without the need for additional solvents.

[0043] In the present application, the concentration of the traditional Chinese medicine composition in the medicine is preferably 0.5 g / g.

[0044] The present application provides the use of the traditional Chinese medicine composition prepared by the preparation method in the preparation of a medicine for preventing and / or treating ovarian dysfunction.

[0045] In the present application, the ovarian dysfunction preferably includes at least one of the following: menstrual cycle disorder, reduced ovarian mass, reduced number of primary and mature follicles, reduced number of corpus luteum, hormone disorder and increased sex hormone receptor. The hormone disorder preferably includes at least one of the following: reduced anti-Mullerian hormone (AMH) and follicle-stimulating hormone (FSH), increased serum testosterone (T) and increased luteinizing hormone (LH). The sex hormone receptor preferably includes CYP19A1 and LHR.

[0046] In the present application, in order to investigate the effect of glucose and lipid metabolism disorder on the ovarian health of women of childbearing age, a mouse model of T2DM with glucose and lipid metabolism disorder and ovarian dysfunction is constructed. Specifically, the mice are fed with high-fat feed for 4 weeks, and then injected with streptozotocin (STZ, 40 mg / kg / d) for 5 consecutive days. The estrous cycle and serum hormone content of the mice are determined, and the ovarian sections are observed. The results show that, compared with the blank control group (Con), the model group mice have estrous cycle disorder, specifically, the estrus period is shortened, and the post-estrus and inter-estrus periods are prolonged; the ovarian mass is reduced, the number of follicles and corpus luteum at all levels is reduced, and the number of atretic follicles is increased, among which the number of corpus luteum and mature follicles is most significantly reduced, and the percentage of mature follicles is also significantly reduced. The above results show that the mouse model has ovarian dysfunction, and a disease animal model is successfully constructed.

[0047] In the embodiment of the present application, in order to further explore the effect of improving ovarian function disorder and the dose-effect relationship of the traditional Chinese medicine composition, low-dose group (3.0 g / kg / d), medium-dose group (6.0 g / kg / d), high-dose group (12.0 g / kg / d) and super high-dose group (24.0 g / kg / d) are set. Compared with the model group, different doses of drug treatment can restore the estrus period to different degrees, among which the estrus period of the SQTL-M group is significantly prolonged, and the estrus cycle is most significantly restored. The serum hormone results show that compared with the model group, the AMH, FSH, T and LH serum levels of each treatment group of the traditional Chinese medicine composition are restored to different degrees. The RT-qPCR results show that each dose group of the traditional Chinese medicine composition has different degrees of recovery on the expression of CYP19A1 and LHR mRNA expression levels. In addition, the traditional Chinese medicine composition also evaluates the appearance of the ovary, the ovarian index and the number of follicles. Except for the super high-dose group, the traditional Chinese medicine composition can improve the reduction of the ovarian quality of the mice to different degrees, and the ovarian index gets basically the same results; the traditional Chinese medicine composition each treatment group of mice ovary can see different degrees of increase of all levels of follicles, the number of atretic follicles is reduced, and the percentage of mature follicles is increased. Among them, the medium-dose group has the best improvement effect.

[0048] The application of the traditional Chinese medicine composition provided by the present application in preparing a medicine for preventing and / or treating ovarian function disorder will be described in detail below in combination with examples, but they cannot be understood as limiting the protection scope of the present application.

[0049] Example 1

[0050] Preparation method of traditional Chinese medicine granules

[0051] 1) Accurately weigh the following traditional Chinese medicine raw materials: American ginseng 9g, Sanchi 3g, Gegen 60g, Huanglian 45g, Huangqi 45g, Jieseng 15g, Ganjiang 15g, Kugua 45g, Zhimu 60g;

[0052] 2) Mix and crush the above weighed traditional Chinese medicine raw materials to obtain traditional Chinese medicine powder, then extract twice with 10 times water for 100 min, concentrate the two times of extraction liquid to 297 mL, cool to room temperature, then add industrial alcohol to the alcohol content of the liquid to be 40% (297 mL of concentrated liquid + 198 mL of industrial alcohol), alcoholize for 24 h, filter with gauze folded four times, concentrate, put in the oven at 60℃ and dry for about 3h, grind to powder to obtain traditional Chinese medicine composition powder;

[0053] 3) The traditional Chinese medicine composition powder is placed in a V-type mixer with 10% to 20% dextrin as a binder and 20% to 30% sucrose powder or bitter taste inclusion excipient β-cyclodextrin as a filler / corrective, and dry mixed for 10 minutes to uniformity. Finally, granulation is performed: dry granulation is adopted, the mixed powder is directly compressed into a thin slice by a roller press, and then broken and granulated, and a microwave-assisted granulation process is preferably adopted: 5% to 8% water is left in the powder, and the water is heated to 60 to 80°C by microwave to promote water vaporization to realize powder bonding, so that granulation can be achieved without adding an external solvent, and a traditional Chinese medicine granule is obtained.

[0054] Example 2

[0055] Application of traditional Chinese medicine compound in treatment of ovarian function disorder

[0056] 1. Materials

[0057] 1.1 Experimental animals

[0058] 4-week-old female C57BL / 6J mice, weighing 20±2 g, were purchased from the Experimental Animal Center of Ningxia Medical University (ethics number). The mice were raised in the Experimental Animal Center of Ningxia Medical University, and the living environment temperature was 20-22°C, the relative humidity was 60%-80%, and the light-dark alternation was 12 hours. The mice could freely obtain food and water. After 1 week of adaptive feeding, mice with normal blood glucose and regular estrous cycles were selected for formal experiments. This study conforms to the animal ethics, and is approved by the Experimental Animal Welfare Ethics Committee of Ningxia Medical University, and conforms to the "Animal Protection, Animal Welfare and Ethical Principles" (IACUC).

[0059] 1.2. Drugs

[0060] Traditional Chinese medicine granules (composed of 9 kinds of Chinese herbal medicine, including American ginseng 9 g, Sanchi 3 g, Gegen 60 g, Huanglian 45 g, Huangqi 45 g, Juecang 15 g, Ganjiang 15 g, Kugua 45 g, Zhimu 60 g). According to the dose-effect relationship of prescription medicine, the low-dose group was administered 3.0 g / kg / d (the crude drug administration dose was 18.018 g / kg); the medium-dose group was administered 6.0 g / kg / d (the crude drug administration dose was 36.036 g / kg), and the above dose groups were configured into 0.5 g / ml solution; the high-dose group was administered 12.0 g / kg / d (the crude drug administration dose was 72.072 g / kg), and the ultra-high-dose group was administered 24.0 g / kg / d (the crude drug administration dose was 144.144 g / kg), and the above dose groups were configured into 2 g / 3 ml solution. STZ (Sigma, #WXBD6414V); Metformin hydrochloride tablets (Shanghai Squibb Pharmaceutical Co. LTD); Insulin (Wanbang Medicine, 22301203); Glucose (DAMAO, 14431-43-7); the above drugs were stored at appropriate temperatures according to the instructions.

[0061] 1.3. Main reagents

[0062] High-fat feed (60% fat, Reasearch Diets, D12492); DNA / RNA / Protein Kit (Omega, #R6734020000C31V007); DNA / RNA / Protein Kit (Omega BIO-TEK, R6734-02); Mouse testosterone (T) enzyme-linked immunosorbent assay kit (JONLN BIO, JL25196), mouse estrogen (E) enzyme-linked immunosorbent assay kit (JONLN BIO, JL11232), mouse follicle-stimulating hormone (FSH) enzyme-linked immunosorbent assay kit (JONLN BIO, JL10239), mouse luteinizing hormone (LH) enzyme-linked immunosorbent assay kit (JONLN BIO, JL10432); Mouse anti-Mullerian hormone (AMH) kit (ELISA, Quanzhou Jiubang Biological Technology Co., Ltd., QZ-13792); TUNEL staining kit (Elabscience, E-CK-A320); Reverse transcription kit (PrimeScriptTM RT Master Mix, TaKaRa, RR036A).

[0063] 1.4. Main instruments

[0064] Roche glucometer (Roche, Switzerland); Roche glucometer strips (Roche, Switzerland); table top high speed centrifuge (Eppendorf, Germany), microplate reader (Multiskan GO, US), Nanodrop 2000 (Thermo, US), electrophoresis apparatus / electrophoresis tank / transmembrane tank (Bio-Rad, US); GelDoc XR chemiluminescence gel imaging system (BioRad Instruments, US); Nikon A1 confocal laser microscope (Nikon, Japan); TissueGnostics whole slide quantitative analysis machine (TissueGnostics, Austria).

[0065] 2. Experimental methods

[0066] 2.1 Establishment and treatment of T2DM with glucose and lipid metabolism disorder and ovarian function disorder mouse model

[0067] Four-week-old female C57BL / 6J mice weighing 20±2g were adaptively fed for 1 week and then randomly divided into a control group (Con) (n=6) and a model group (n=42). The blank control group was established, and the control group mice were fed with regular feed for 4w (free drinking), and then intraperitoneally injected with 40mg / kg / d normal saline for 5d in succession, and continued to be fed with regular feed until sampling; the model group mice were first given high-fat feed for 4w (free drinking), and then intraperitoneally injected with 40mg / kg / d streptozotocin (STZ) for 5d in succession; 10d after the model group mice were intraperitoneally injected with STZ, the model group mice were randomly divided into a model group (Model), a positive drug control group (MET), a prescription low-dose group (SQTL-L, 3.0g / kg / d), a prescription medium-dose group (SQTL-M, 6.0g / kg / d), a prescription high-dose group (SQTL-H, 12.0g / kg / d), and a prescription ultrahigh-dose group (SQTL-UH, 24.0g / kg / d) (n=6) after the success of the model was confirmed. During the treatment, all mice were fed with ordinary feed, and the corresponding dose of traditional Chinese medicine was given to each treatment group by gavage. After 8w of treatment, the estrous cycle was monitored for 1w before the end of treatment.

[0068] 2.2 Determination of estrous cycle by vaginal smear method

[0069] Vaginal smears were collected daily at 4:00 pm for 7 days from day 4 to day 10 after intraperitoneal injection of STZ and 7 days before sample collection. A small amount of 0.9% saline was sucked into the vagina with a Pasteur pipette, the pipette was inserted into the mouse vagina for 5 mm, and the vaginal secretion was placed on a glass slide. The estrous cycle was observed under a light microscope and recorded. It was naturally dried, a suitable amount of 95% alcohol was dropped for fixation, and after drying, it was used for ultra-fast-non-toxic modified Pasteur stain kit staining. Nucleated epithelial cells were blue-green, anucleated keratinized squamous cells were pink, leukocytes were blue-purple, and mucus was light blue or pink. [34,35] .

[0070] 2.3 Blood and ovary collection

[0071] The mouse body weight was weighed before sampling, the mouse was anesthetized with sodium pentobarbital (3%, 1 ml / kg), blood was collected with EDTA as an anticoagulant, and the blood was centrifuged at 1000 x g for 20 minutes at 2-8°C within 30 minutes after collection. The supernatant was collected and stored at -80°C to avoid repeated freezing and storage. The bilateral ovaries of the mouse were collected, and the wet weight of the bilateral ovaries of the mouse was weighed. The right ovary was fixed in 4% paraformaldehyde solution, and the left ovary and serum were stored at -80°C. The ovary index was calculated according to formula I:

[0072] Ovary index (%) = bilateral ovary weight (g) / mouse body weight (g) x 100% formula I.

[0073] 2.4 Serum hormone concentration analysis

[0074] The mouse heart blood was centrifuged at 4°C and 4000 rpm for 10 min, the supernatant was taken, and the T, AMH, FSH, E, and LH levels in the serum were determined according to the ELISA kit instructions. The absorbance value was measured at 450 nm wavelength using a microplate reader (Thermo MULTISKAN GO), and the hormone concentration was calculated.

[0075] 2.5 Ovary H&E staining

[0076] After 4% paraformaldehyde room temperature fixation for 24 h, the ovarian tissues were dehydrated in different concentrations of ethanol, paraffin-embedded, and sectioned into 4 pm sections. H&E staining was performed. The pathological morphological changes of the ovaries were observed under a BX-51 upright fluorescence microscope of Japan OL YMPUS company, and the number of follicles at each stage was counted. According to a previous study, follicles were divided into five stages: primordial follicle, primary follicle, secondary follicle, antral follicle and atretic follicle. Primordial follicles were surrounded by a single layer of flat granulosa cells, primary follicles were surrounded by a single layer of cubic granulosa cells, secondary follicles had at least two layers of granulosa cells, lacked follicular lumen, antral follicles contained multiple layers of granulosa cells and follicular lumen, and atretic follicles were composed of atrophied oocytes and abnormal granulosa cells. Primary follicles, secondary follicles, mature follicles and atretic follicles were observed under a 20-fold magnifying lens. To avoid repeated counting, only follicles with visible oocyte nuclei were counted.

[0077] 2.6 TUNEL staining

[0078] Paraffin sections were taken and apoptosis in the ovaries was determined according to the One-step TUNEL In Situ Apoptosis Kit (Elabscience, E-CK-A320) kit instructions. Nikon Al confocal laser microscope was used to collect TUNEL staining images.

[0079] 2.7 RT-qPCR detection of CYP19A1, FSHR and LHR mRNA expression levels

[0080] RNA was extracted using a DNA / RNA / Protein Kit (Omega, R6734-02) and reverse transcribed to obtain cDNA. According to the PrimeScript™ RT Master Mix (TaKaRa, RR036A) instructions, a 20 pl reaction system was prepared and RT-qPCR was performed. The specific reaction system is shown in Table 1. The primer sequences used in this experiment are shown in Table 2. PCR-7500 Fast instrument was used, and the program settings were as follows: pre-denaturation - 95°C, 30s, PCR reaction - 95°C, 3s, 60°C-30s, the above settings were performed for 40 cycles, the samples were put on the machine and the program was run.

[0081] Table 1 RT-PCR system (20 pl)

[0082]

[0083]

[0084] Table 2 Primer sequences

[0085]

[0086] 2.8 Statistical Analysis

[0087] The obtained data were statistically analyzed using Prism 9.0 statistical analysis software. Measurement data are expressed as mean ± standard error. Two-way ANOVA was used to compare the means of multiple samples. One-way ANOVA was used for pre-sampling body weight, ovarian index, follicle count, serum hormone levels, and qPCR. A p-value < 0.05 was considered statistically significant between groups.

[0088] 3. Results

[0089] 3.1 Constructing a mouse model of impaired ovarian function

[0090] First, we verified the changes in ovarian morphology and function in the model mice. We monitored the estrous cycle of the mice during modeling, such as... Figure 1 As shown in Figure H, compared with the Con group, the Model group mice exhibited disordered estrous cycles, specifically a shortened estrous period and a prolonged metestinal and interestrous period. After euthanizing the mice and preserving their ovaries, the ovaries of both sides were weighed. Compared with the Con group, the ovarian mass of the Model group mice was significantly reduced. Figure 1 (A). H&E staining and follicle counting results showed ( Figure 1 In the BH group, compared with the Con group, the Model group mice showed a decrease in the number of follicles and corpora lutea at all stages, and an increase in atretic follicles. The most significant decreases were in the number of corpora lutea and mature follicles, and the percentage of mature follicles was also significantly reduced. These results collectively indicate that the mouse model exhibits ovarian dysfunction.

[0091] 3.2 Effects of Traditional Chinese Medicine Compound on Estrogenic Cycle, Serum Hormones, and Sex Hormone Receptors in Model Mice

[0092] For seven consecutive days before the end of treatment, the estrous cycles of mice in each group were monitored. After induction with STZ and a high-fat diet, the estrous cycles of mice in the Model group were disordered, characterized by a shortened estrous period and a prolonged metestinal and interestrous period. Figure 2 (E, F, and L); different doses of SQTL treatment could restore the estrous cycle to varying degrees, among which the SQTL-M group mice showed a significant prolongation of the estrous cycle and the most significant restoration of the estrous cycle (E, F, and L); Figure 2 (EL). Serum hormone results showed ( Figure 2 In the MET / SQTL treatment group, compared with the Con group, the Model group mice showed significantly decreased AMH and FSH levels (p < 0.05), and significantly increased LH and T levels (p < 0.05), with no significant difference in E levels among the groups. Compared with the Model group, the serum levels of AMH, T, LH, and FSH in each MET and SQTL treatment group showed varying degrees of recovery. RT-qPCR results showed ( Figure 2Compared with the Con group, the expression levels of CYP19A1 and LHR mRNA in the Model group were significantly increased, and SQTL at each dose restored the expression of the above factors to varying degrees. There was no significant difference in the expression of FSHR mRNA among the groups.

[0093] 3.3 Effects of traditional Chinese medicine compound on the appearance of ovaries, ovarian index, and follicle number in model mice

[0094] The ovaries of mice were photographed, weighed, and the ovarian index was calculated. Figure 3 Compared with the Con group, the ovarian mass of the Model group was significantly reduced, and all treatment groups except SQTL-UH improved the ovarian mass to varying degrees. The ovarian index showed basically the same results, but there was no statistical difference among the groups. H&E staining of mouse ovaries Figure 3 J) showed that the ovaries of the Con group contained various follicles (primary follicles, secondary follicles, mature follicles, and atretic follicles), and the structure of each follicle was complete, with clear cell morphology. The ovaries of the Model group contained fewer follicles, and the ovaries of the treatment groups contained varying degrees of follicles. Follicle counting was performed on mice in each group Figure 3 D-H), compared with the Con group, the number of primary follicles and mature follicles in the Model group was significantly reduced, and the percentage of mature follicles was significantly reduced, with statistical significance (p<0.05). The number of secondary follicles and corpus luteum was reduced, and the number of atretic follicles was increased, but there was no statistical difference. After SQTL treatment, the number of follicles at each level increased, and the number of atretic follicles decreased, with the best improvement in the SQTL-M group, but there was no statistical difference among the treatment groups. In addition, the percentage of mature follicles increased Figure 3 I) after treatment, and the increase in the SQTL-M group was the most significant.

[0095] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A traditional Chinese medicine composition, characterized in that, The following Chinese medicinal materials are included in parts by weight: American ginseng 8-10 parts, Panax notoginseng 2-4 parts, Pueraria lobata 55-65 parts, Coptis chinensis 42-48 parts, Scutellaria baicalensis 42-48 parts, Bombyx mori 14-16 parts, dried ginger 14-16 parts, bitter melon 42-48 parts, and Anemarrhena asphodeloides 55-65 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The following Chinese medicinal materials are included in the following weight proportions: American ginseng 9 parts, Panax notoginseng 3 parts, kudzu root 60 parts, Coptis chinensis 45 parts, Scutellaria baicalensis 45 parts, Bombyx mori 15 parts, dried ginger 15 parts, bitter melon 45 parts, and Anemarrhena asphodeloides 60 parts.

3. The method for preparing the traditional Chinese medicine composition according to claim 1 or 2, characterized in that, Includes the following steps: American ginseng, Panax notoginseng, kudzu root, Coptis chinensis, Scutellaria baicalensis, Bombyx batryticatus, dried ginger, bitter melon, and Anemarrhena asphodeloides were extracted with water at 70–90°C to obtain an extract. The extract was concentrated and purified with a 35%–45% (v / v) alcohol solution. The supernatant was collected, and the alcohol solvent was removed to obtain the traditional Chinese medicine composition.

4. The preparation method according to claim 3, characterized in that, When performing water extraction or alcohol extraction, the material-to-liquid ratio is 1:8 to 1:12; The water extraction or alcohol extraction time is 1 to 2 hours, and the water extraction or alcohol extraction is performed 2 to 3 times. The ultrasonic-assisted extraction time is 30–60 min; the ultrasonic-assisted extraction frequency is 40–60 kHz. The concentration includes vacuum concentration, which is concentrated to obtain a clear extract with a relative density of 1.05-1.15 at 60°C; During the alcohol purification process, the working volume percentage of alcohol is 70% to 80%.

5. The use of the traditional Chinese medicine composition according to claim 1 or the traditional Chinese medicine composition prepared by the preparation method according to claim 3 or 4 in the preparation of a drug for the prevention and / or treatment of ovarian dysfunction.

6. The application according to claim 5, characterized in that, The ovarian dysfunction includes at least one of the following: estrous cycle disorder, reduced ovarian quality, decreased number of primary and mature follicles, decreased number of corpora lutea, hormonal imbalance, and elevated sex hormone receptor levels.

7. The application according to claim 6, characterized in that, The hormonal imbalance includes at least one of the following: decreased anti-Müller hormone, increased serum testosterone, and increased luteinizing hormone.

8. The application according to claim 5, characterized in that, In the drug, the concentration of the traditional Chinese medicine composition is 0.5 g / g or higher.

9. A drug for treating ovarian dysfunction, characterized in that, The active ingredients include the traditional Chinese medicine composition according to claim 1 or 2 or the traditional Chinese medicine composition prepared by the preparation method according to claim 3 or 4.

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